The present invention relates in general to SdrG, a fibrinogen-binding bacterial adhesin, and in particular to the use of SdrG or its binding region as an anti-coagulation agent by virtue of its ability to inhibit thrombin-induced fibrin clot formation by interfering with the release of fibrinopeptide B. In addition, the invention relates to methods and compositions utilizing SdrG or its binding region for treating or preventing thrombin-induced coagulation and conditions associated therewith.
Coagulase-negative staphylococci (CNS) are important opportunistic pathogens that are particularly associated with foreign body infections in humans. Staphylococcus epidermidis is the most common pathogenic species of CNS and accounts for 74-92% of the infections caused by this group of staphylococci (1). The molecular pathogenesis of most infections is complex and involves multiple microbial factors and host components, but is generally initiated by the adherence of the microbe to host tissues. Bacterial adherence involves specific surface components called adhesins, and bacterial pathogens, such as staphylococci that live in the extracellular space of the host, target extracellular matrix (ECM) components, including fibrinogen (Fg) and fibronectin, for adherence and colonization. This process is mediated by a sub-family of adhesins that have been termed MSCRAMM(copyright)s (microbial surface components recognizing adhesive matrix molecules) (2). Staphylococcus aureus expresses multiple MSCRAMM(copyright)s of which several have been characterized in some detail (For a recent review see Ref. 3), and various MSCRAMM(copyright)s have been the subject of U.S. Patents, including fibronectin binding proteins such as disclosed in U.S. Pat. Nos. 5,175,096; 5,320,951; 5,416,021; 5,440,014; 5,571,514; 5,652,217; 5,707,702; 5,789,549; 5,840,846; 5,980,908; and 6,086,895; fibrinogen binding proteins such as disclosed in U.S. Pat. Nos. 6,008,341 and 6,177,084; and collagen binding proteins as disclosed in U.S. Pat. Nos. 5,851,794 and 6,288,214; all of these patents incorporated herein by reference. In addition, other information concerning SdrG and other MSCRAMM(copyright)s can be found in U.S. Ser. No. 09/810,428, filed Mar. 19, 2001, incorporated herein by reference; and U.S. Ser. No. 09/386,962, filed Aug. 31, 1999, incorporated herein by reference.
In addition to S. epidermidis, S. aureus also causes serious foreign body infections. S. aureus appears to adhere to the biomaterial through an indirect mechanism. Upon implantation, the foreign body rapidly becomes coated with host proteins derived primarily from plasma with Fg being a dominant component. S. aureus appears to adhere to the absorbed proteins rather than to the biomaterial itself using adhesins of the MSCRAMM(copyright) family (4,5). At least four of the S. aureus MSCRAMM(copyright)s recognize Fg. Two of these MSCRAMM(copyright)s, clumping factor A and B (ClfA , ClfB ), have Fg-binding A-regions followed by a long segment of Ser-Asp (SD) dipeptide repeats. The other two Fg-binding MSCRAMM(copyright)s, contain a similar ligand binding A-region followed by a fibronectin binding motif that is repeated 5 times (6). Because the fibronectin binding activity was identified first, these two MSCRAMM(copyright)s are known as fibronectin binding protein A and B (FnbpA and FnbpB) (7,8). Studies have demonstrated the importance of ClfA and ClfB in the adherence of S. aureus to plasma-coated biomaterials. S. aureus mutants deficient in one or both of these MSCRAMM(copyright)s exhibited an impaired ability to adhere to plasma-coated catheters in vivo or ex vivo (9,10).
For S. epidermidis, adherence to foreign bodies appears to involve both specific and non-specific processes. The bacteria may initially associate directly with the foreign body through non-specific interactions, while the later stages of adherence may involve more specific interactions between bacterial adhesins and host ligands. S. epidermidis expresses polysaccharide adhesins including PS/A and PIA, which are encoded by the ica locus (11, 12). In addition, the present inventors (13) and others (14) have recently shown that S. epidermidis contains surface proteins structurally related to S. aureus MSCRAMM(copyright)s. Two of these S. epidermidis proteins, called SdrF and SdrG, have features typical of Gram-positive bacterial proteins that are anchored to the cell wall. Both proteins show significant amino acid sequence homology to ClfA and ClfB from S. aureus including an xcx9c500 amino acid long A region, a SD dipeptide repeat region and features required for cell wall anchoring, including a LPXTG (SEQ ID NO:1) motif (FIG. 1A). Recent studies by Pei, et al. suggest that another S. epidermidis protein, called Fbe, can bind Fg and, much like SdrG, specifically recognizes the Bxcex2 chain of this molecule (15). However, this reference does not disclose or suggest the specific binding site for the Fbe protein on fibrinogen and thus does not disclose or suggest that the binding site for this protein would be related to of affect in any manner the binding site for thrombin on fibrinogen.
Of these proteins from S. epidermidis, SdrG is of particular interest for its ability to bind Fg. Fg is known to play a critical role in the formation of blood clots, although previously the precise binding site of SdrG to Fg has not been localized with specificity. Accordingly, because the precise binding site for SdrG in the Fg Bxcex2 chain has not been localized, it has not been previously been associated with the thrombin cleavage site on fibrinogen and thus it has not previously been recognized or suggested that SdrG might be useful in inhibiting the thrombin-induced cleavage of fibrinogen and the thrombin-induced process of clot formation.
In general, the blood clots generated by Fg, e.g. through its cleavage by thrombin to form fibrin and start the process of blood coagulation, are beneficial in the normal wound healing process. However, abnormal clots caused by the cleavage of Fg can lead to thrombosis, a condition where a clot develops in the circulatory system. Thrombosis is an extremely dangerous condition and may produce ischemic necrosis of the tissue supplied by the artery, e.g., myocardial infarction due to thrombosis of a coronary artery, or stroke due to thrombosis of a cerebral artery. In addition to the above, venous thrombosis may cause the tissues drained by the vein to become edematous and inflamed, and thrombosis of a deep vein may result in a pulmonary embolism. Still other problems result in sickle-cell patients wherein the malformed xe2x80x9csickle cellsxe2x80x9d can also lead to a sickle-cell crisis state in which coagulation reaches dang once again result in serious injury or even death.
Generally, anticoagulant agents such as heparin and its derivatives are used to treat thrombosis and to prevent or reduce coagulation when desirable such as in the case of myocardial infarction and the other conditions discussed above. Heparin works by inhibiting thrombin generation and in antagonizing thrombin""s action. However, the use of heparin has distinct problems which have yet to be overcome. One disadvantage associated with heparin is that it can only be administered parenterally. Another serious disadvantage is major bleeding occurs in 1% to 33% of patients who receive various forms of heparin therapy. In fact, purpura, ecchymoses, hematomas, gastrointestinal hemorrhage, hematuria, and retroperitoneal bleeding are regularly encountered complications of heparin therapy. In addition to the above complications, thrombocytopenia occurs in 1% to 5% of patients receiving heparin.
Accordingly, there is thus a distinct and growing need to provide alternatives to heparin as anti-coagulation agent which do not suffer from all of the above-mentioned side effects or disadvantages. One such alternative is the use of snake venom products including ancrod, an xcex1-fibrinogenase isolated from Calloselasma rhodostoma (Malayan Pit viper). However, ancrod appears to release only FpA and leads to the formation of an unstable fibrin clot (Bell 1997). Moreover, because this defibrinating enzyme cleaves FpA and not FpB from Fg, it forms a clot that is very sensitive to endogenous fibrinolysis, and additionally activates plasminogen further contributing to fibrinolysis (Pizzo, Schwartz et al. 1972; Carr 1975; Bell 1997). Hypofibrinogenemia, i.e., the reduction of Fg in the blood, must be sustained by administering a treatment, the plasma Fg rises and returns to normal levels in days (Bell, Bolton et al. 1968). The limited clinical experience indicates that which defibrination may be achieved with ancrod, the elaboration of neutralizing antibodies with repeated injections of ancrod leads to resistance (see, e.g., Pitney, Holt et al. 1969; Pitney and Regoeczi 1970), (Vinazzer 1973; Sapru, Moza et al. 1975) .
In short, there is a distinct and acute need to provide a safe and effective alternative to the use of heparin in achieving the inhibition in blood coagulation in cases wherein such inhibition may be crucial in saving the life of a human or animal patient.
It is thus an object of the present invention to provide safe and effective alternatives to the use of heparin in achieving therapeutic anti-coagulant treatment in human and animal patients.
It is further an object of the present invention to provide methods of utilizing fibrinogen-binding proteins from S. epidermidis in the prevention or treatment of thrombin-induced coagulation in human or animal patients.
It is further an object of the present invention to provide methods of utilizing fibrinogen-binding proteins from S. epidermidis in order to reduce or prevent thrombin-induced coagulation and to enhance the dissolution of blood clots in human or animal patients.
It is another object of the invention to provide therapeutic compositions based on fibrinogen-binding proteins from S. epidermidis which bind to the Bxcex2 chain of fibrinogen which are useful in preventing or treating thrombin-induced coagulation in human or animal patients in need thereof.
It is still further an object of the present invention to develop compositions from fibrinogen-binding proteins from S. epidermidis which bind to the Bxcex2 chain of fibrinogen, and which can block the thrombin binding site on fibrinogen so as to be useful in methods of preventing cleavage of fibrinogen by thrombin and inhibiting the release of fibrinopeptide B from fibrinogen.
These and other objects are provided by virtue of the present invention which comprises compositions and methods which utilize the SdrG protein from S. epidermidis, and other proteins which bind to the Bxcex2 chain of fibrinogen such as the A region of SdrG and the Fbe protein, in order to treat or prevent thrombin-induced coagulation in human or animal patients. In addition, the invention comprises methods of administering SdrG so as to treat or prevent a wide variety of conditions wherein blood coagulation can be dangerous or even life-threatening coagulation, including venous thrombosis, myocardial infarction and sickle cell crisis episodes. The invention utilizes the ability of SdrG to inhibit thrombin-induced fibrin clot formation by inhibiting thrombin binding to fibrinogen and interfering with the release of fibrinopeptide B, and therapeutic compositions containing an effective amount of SdrG can thus be used as effective anti-coagulation agents. The SdrG compounds and compositions of the present invention may also be used to reduce the concentration of plasma fibrinogen in a patient""s blood when so desired.
These embodiments and other alternatives and modifications within the spirit and scope of the disclosed invention are described in, or will become readily apparent from, reference to the detailed description of the preferred embodiments provided herein below.